RESUMO
Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor-binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain-containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.
Assuntos
RNA Polimerase I , Precursores de RNA , Humanos , Animais , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , DNARESUMO
DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.
Assuntos
Cromatina/metabolismo , Cromossomos/genética , DNA Topoisomerases Tipo II/genética , Histonas/genética , Adenosina Trifosfatases/metabolismo , Animais , Extratos Celulares/química , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Feminino , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Oócitos/química , Oócitos/metabolismo , Fuso Acromático/genética , Fuso Acromático/patologia , Fuso Acromático/ultraestrutura , Xenopus laevisRESUMO
In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.
Assuntos
RNA Polimerase III/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Estabilidade Enzimática , Células HeLa , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Subunidades Proteicas , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Condensin and cohesin, both members of the structural maintenance of chromosome (SMC) family, contribute to the regulation and structure of chromatin. Recent work has shown both condensin and cohesin extrude DNA loops and most likely work via a conserved mechanism. This review focuses on condensin complexes, highlighting recent in vitro work characterising DNA loop formation and protein structure. We discuss similarities between condensin and cohesin complexes to derive a possible mechanistic model, as well as discuss differences that exist between the different condensin isoforms found in higher eukaryotes.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Ligação a DNA/química , DNA/química , Complexos Multiproteicos/química , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/química , Chaetomium/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Microscopia Crioeletrônica , Dimerização , Regulação Fúngica da Expressão Gênica , Humanos , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.
Assuntos
Capuzes de RNA/genética , Infecções por Vírus de RNA/genética , Proteínas Recombinantes de Fusão/genética , Regiões 5' não Traduzidas/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Cães , Humanos , Vírus da Influenza A/metabolismo , Camundongos , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Fases de Leitura Aberta/genética , Capuzes de RNA/metabolismo , Infecções por Vírus de RNA/metabolismo , Vírus de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica/genética , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula/métodosRESUMO
How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.
Assuntos
Elementos Alu/fisiologia , Histonas/metabolismo , Fatores de Transcrição TFIII/metabolismo , Acetilação , Elementos Alu/genética , Linhagem Celular , Cromatina/metabolismo , Cromatina/fisiologia , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Histonas/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional , RNA Polimerase III/metabolismo , Fatores de Transcrição TFIII/genética , Transcrição Gênica/genéticaRESUMO
RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4-4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation.
Assuntos
RNA Polimerase III/metabolismo , RNA Polimerase III/ultraestrutura , Iniciação da Transcrição Genética , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase I/química , RNA Polimerase II/química , RNA Polimerase III/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Moldes Genéticos , Fator de Transcrição TFIIIB/química , Fator de Transcrição TFIIIB/metabolismo , Fator de Transcrição TFIIIB/ultraestrutura , Fatores de Transcrição TFII/químicaRESUMO
Here, we discuss the role of Brf2, an RNA Polymerase III core transcription factor, as a master switch of the oxidative stress response. We highlight the interplay of Brf2 with the Nrf2/Keap1 pathway, as well as the role of Brf2 in cancer and other possible regulations.
Assuntos
Neoplasias/genética , Estresse Oxidativo/genética , RNA Polimerase III/metabolismo , Fator de Transcrição TFIIIB/metabolismo , Transcrição Gênica , Humanos , Neoplasias/metabolismoRESUMO
TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.
Assuntos
Oxirredução , Fator de Transcrição TFIIIB/química , Fator de Transcrição TFIIIB/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , RNA Polimerase III/metabolismo , Saccharomyces cerevisiae , Alinhamento de Sequência , Transdução de SinaisRESUMO
RNA polymerase I and III are responsible for the bulk of nuclear transcription in actively growing cells and their activity impacts the cellular biosynthetic capacity. As a consequence, RNA polymerase I and III deregulation has been directly linked to cancer development. The complexity of RNA polymerase I and III transcription apparatuses has hampered their structural characterization. However, in the last decade tremendous progresses have been made, providing insights into the molecular and functional architecture of these multi-subunit transcriptional machineries. Here we summarize the available structural data on RNA polymerase I and III, including specific transcription factors and global regulators. Despite the overall scarcity of detailed structural data, the recent advances in the structural biology of RNA polymerase I and III represent the first step towards a comprehensive understanding of the molecular mechanism underlying RNA polymerase I and III transcription. This article is part of a Special Issue entitled: Transcription by Odd Pols.
Assuntos
RNA Polimerase III/química , RNA Polimerase I/química , Transcrição Gênica , Animais , Humanos , RNA Polimerase I/metabolismo , RNA Polimerase III/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.
Assuntos
Histona Desacetilases/química , Histona Desacetilases/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Ácido Aspártico/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single alpha/beta domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.
Assuntos
Inibidores de Histona Desacetilases , Histona Desacetilases/química , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Domínio Catalítico , Linhagem Celular Tumoral , Dicroísmo Circular , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Histona Desacetilases/genética , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Potássio/metabolismo , Conformação Proteica , Interferência de RNA , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Zinco/metabolismoRESUMO
Transfer of the tumor-inducing plasmid in Agrobacterium tumefaciens is controlled by a quorum-sensing system whose main components are the transcriptional regulator TraR and its autoinducer. This system allows bacteria to synchronize infection of the host plant when a "quorum" of cells has been reached. TraM is an A. tumefaciens protein involved in the regulation of this system because it binds to TraR and prevents it from binding DNA. As a first step to understanding the molecular basis for the regulation of TraR by TraM, we have determined the crystal structure of TraM at 1.65 A resolution. This protein is packed as a dimer, with each monomer consisting mainly of two antiparallel alpha helices. Monomers are tightly associated, with a large hydrophobic area buried upon dimerization. Secondly, we characterized the TraR-TraM complex in vitro. TraM (11.4 kDa, monomer molecular mass) binds tightly TraR (27 kDa, monomer molecular mass) forming a stable oligomeric complex that likely accounts for two TraR and two TraM dimers.
Assuntos
Proteínas de Bactérias/química , Agrobacterium tumefaciens/metabolismo , Cromatografia , Cristalografia por Raios X , Dimerização , Elétrons , Cinética , Luz , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Espalhamento de RadiaçãoRESUMO
The aim of this study was to investigate the mechanism of activation of human heparanase, a key player in heparan sulfate degradation, thought to be involved in normal and pathologic cell migration processes. Active heparanase arises as a product of a series of proteolytic processing events. Upon removal of the signal peptide, the resulting, poorly active 65 kDa species undergoes the excision of an intervening 6 kDa fragment generating an 8 kDa polypeptide and a 50 kDa polypeptide, forming the fully active heterodimer. By engineering of tobacco etch virus protease cleavage sites at the N- and C-terminal junctions of the 6 kDa fragment, we were able to reproduce the proteolytic activation of heparanase in vitro using purified components, showing that cleavage at both sites leads to activation in the absence of additional factors. On the basis of multiple-sequence alignment of the N-terminal fragment, we conclude that the first beta/alpha/beta element of the postulated TIM barrel fold is contributed by the 8 kDa subunit and that the excised 6 kDa fragment connects the second beta-strand and the second alpha-helix of the barrel. Substituting the 6 kDa fragment with the topologically equivalent loop from Hirudinaria manillensis hyaluronidase or connecting the 8 and 50 kDa fragments with a spacer of three glycine-serine pairs resulted in constitutively active, single-chain heparanases which were comparable to the processed, heterodimeric enzyme with regard to specific activity, chromatographic profile of hydrolysis products, complete inhibition at NaCl concentrations above 600 mM, a pH optimum of pH approximately 5, and inhibition by heparin with IC(50)s of 0.9-1.5 ng/microL. We conclude that (1) the heparanase heterodimer (alpha/beta)(8)-TIM barrel fold is contributed by both 8 and 50 kDa subunits with the 6 kDa connecting fragment leading to inhibition of heparanase by possibly obstructing access to the active site, (2) proteolytic excision of the 6 kDa fragment is necessary and sufficient for heparanase activation, and (3) our findings open the way to the production of recombinant, constitutively active single-chain heparanase for structural studies and for the identification of inhibitors.
Assuntos
Glucuronidase/química , Glucuronidase/genética , Mutagênese Sítio-Dirigida , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Clonagem Molecular , Sequência Consenso , Endopeptidases/química , Endopeptidases/genética , Ativação Enzimática/genética , Vetores Genéticos , Humanos , Hidrólise , Dados de Sequência Molecular , Potyvirus/enzimologia , Potyvirus/genética , Dobramento de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Spodoptera/genética , Transfecção , Triose-Fosfato Isomerase/químicaRESUMO
The quorum sensing system allows bacteria to sense their cell density and initiate an altered pattern of gene expression after a sufficient quorum of cells has accumulated. In Agrobacterium tumefaciens, quorum sensing controls conjugal transfer of the tumour- inducing plasmid, responsible for plant crown gall disease. The core components of this system are the transcriptional regulator TraR and its inducing ligand N-(3-oxo-octanoyl)-L-homoserine lactone. This complex binds DNA and activates gene expression. We have determined the crystal structure of TraR in complex with its autoinducer and target DNA (PDB code 1h0m). The protein is dimeric, with each monomer composed of an N-terminal domain, which binds the ligand in an enclosed cavity far from the dimerization region, and a C-terminal domain, which binds DNA via a helix-turn-helix motif. The structure reveals an asymmetric homodimer, with one monomer longer than the other. The N-terminal domain resembles GAF/PAS domains, normally fused to catalytic signalling domains. In TraR, the gene fusion is between a GAF/PAS domain and a DNA-binding domain, resulting in a specific transcriptional regulator involved in quorum sensing.